JP2009143882A - Soya-cerebroside i derivative having tyrosinase-inhibiting activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a soya-cerebroside I derivative having weak side effects and excellent tyrosinase-inhibiting activity. <P>SOLUTION: The soya-cerebroside I derivative having the tyrosinase-inhibiting activity has a structure in which the 3-position of cyanidin having activity for preventing oxidation is bonded to the 6-position of the glucose of the soya-cerebroside I, and cysteine is bonded to the glucose. The oxidation of a fatty acid site of the soya-cerebroside I is prevented by the cyanidin to stabilize the structure. The fatty acid site and the cyanidin site of the soya-cerebroside I act on the tyrosinase. The derivative is obtained by adding Bacillus natto to a pulverized product of longan and a pulverized product of soybean, fermenting the resultant mixture, and alkali-reducing the fermented product. By the fermentation by the Bacillus natto, the 3-position of the cyanidin ester-bonds to the 6-position of the glucose of the soya-cerebroside I, and the cysteine bonds. When the fermented product is alkali-reduced, the structure is stabilized. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a soya cerebroside I derivative having a tyrosinase inhibitory action.

One of the obstacles caused by ultraviolet rays is sunburn, spots and skin cancer. Among them, the stain is caused by the activation of tyrosinase in the skin and the tyrosine being changed to melanin, and there is a demand for improvement of the stain in beauty and health.

Spots caused by ultraviolet rays may damage skin health and develop into skin cancer.Preventing the generation of spots is also necessary to maintain health. Cosmetics and health with various whitening effects Supplements have been developed in pursuit of whitening effects.

Examples of cosmetic raw materials that have promoted a whitening action include materials having excellent antioxidant action such as vitamin C, vitamin E, tocotrienol, hydrogen peroxide, catechol, and arbutin. On the other hand, there is a substance that suppresses melanin production by inhibiting an enzyme called tyrosinase that promotes the production of melanin. Of these, a substance having a tyrosinase inhibitory action is more effective because it directly suppresses melanin production.

Many inventions relating to substances that inhibit tyrosinase production and whitening have already been recognized. First, there is a whitening agent characterized by containing bakuchiol as an active ingredient (see, for example, Patent Document 1). However, this bakuchiol is derived from bovine bone lipids and has a safety problem due to the risk of mad cow disease.

There is an invention relating to a tyrosinase inhibitor characterized by containing an extract from the above-ground part of a plant belonging to South Lealanicept as an active ingredient (see, for example, Patent Document 2). However, the active ingredient has not been identified and the structure has not been identified.

There is an invention relating to a tyrosinase inhibitor containing as an active ingredient an extract obtained by extracting tamarind husk with water, a hydrophilic organic solvent or a mixture thereof (see, for example, Patent Document 3). Similarly, the active ingredient has not been identified and the structure has not been identified.

There is an invention relating to a tyrosinase inhibitor characterized by containing a spiro compound (see, for example, Patent Document 3). However, it is a chemically synthesized compound and has side effects, and its use as an industrial cosmetic is limited.

In the invention relating to tyrosinase inhibitors, whitening cosmetics and anti-discoloration agents, it is Erika extract. Erica is an azalea plant also called heather, but this extract does not specify the active ingredient, the structure of the ingredient has not been analyzed, and its function is mild.

Thus, about the extract derived from a plant, the effect | action is not clear, the useful component has not been identified, and the effect | action is light. On the other hand, the action of sulfur compounds typified by colloidal sulfuric acid and glutathione is not clear, and there is an irritating odor due to sulfur, so its use is limited.

In addition, ascorbic acids, hydrogen peroxide, hydroquinone, catechol and the like have been used to lightly bleach the produced melanin.

Ascorbic acid is easily oxidized, discolors and has a problem of quality. In addition, hydrogen peroxide solution is poor in stability and has a problem in safety in use.

Glutathione and colloidal sulfur have a problem in that they tend to cause discoloration and denaturation. Hydroquinones and catechols have problems in safety in use such as skin irritation and allergic properties. The use of these whitening ingredients in products such as cosmetics is limited, and no whitening ingredients and extracts that are sufficiently satisfactory have yet been obtained.
Japanese Patent No. 3900388 Japanese Patent No. 3658548 Japanese Patent No. 3340878 Japanese Patent No. 3331028

Glutathione and colloidal sulfur are problematic in that they tend to denature and discolor. Hydroquinone and catechol have problems in use safety such as skin irritation and allergic properties.

On the other hand, there is a problem that although the safety of a naturally derived substance is high, its effect is mild.

Therefore, a natural product-derived substance that exhibits a tyrosinase-suppressing action with weak side effects and an excellent anti-inflammatory effect is desired.

The present invention has been made paying attention to the problems existing in the prior art as described above. The object is to provide a soya cerebroside I derivative which has weak side effects and exhibits an excellent tyrosinase inhibitory action.

In order to achieve the above object, the invention described in claim 1 relates to a soya cerebroside I derivative represented by the following formula (1) having a tyrosinase inhibitory action.

Since this invention is comprised as mentioned above, there exist the following effects.

According to the soya cerebroside I derivative obtained by subjecting fermented material obtained by adding natto to fermented natto seeds and fermented soybeans according to claim 1 to alkali reduction treatment, the side effect is weak and excellent tyrosinase Inhibiting action is exerted.

Hereinafter, the best mode for carrying out the present invention will be described in detail.

The soya cerebroside I derivative represented by the following formula (1) having a tyrosinase inhibitory action obtained by alkali reduction treatment of fermented products obtained by adding natto bacteria to fermented pulverized longan seeds and fermented soybeans will be described. .

In the first place, soya cerebroside I is a kind of lipid derivative biosynthesized by plants and microorganisms, and is characterized by high fat solubility.

The English name is Soya-Cerebroside I, the molecular weight is 714.02, and in the chemical formula, C is 40, H is 75, N is 1 and O is 2.

The chemical name of soya cerebroside I is 2-hydroxy-N-[(4E, 8E) -3-hydroxy-1-[(2R, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6 -(Hydroxymethyl) oxan-2-yl] oxyoctadeca-4,8-dien-2-yl] hexadecanamide.

The soya cerebroside I derivative here means that, as shown in the above formula (1), the 6-position of glucose of soya cerebroside I and the 3-position of cyanidin are bonded, and further, the 2-, 3- and 4-positions of glucose are combined. , L-type cysteine is an ester-linked structure.

Cyanidin has the English name cyanidin or cyanidine. The chemical formula is C15H11O6 and 3,5,7,3 ', 4'-pentahydroxyflavylium. The molecular weight is 287.24.

Soya cerebroside I has a fatty acid side chain bonded thereto, and is easily oxidized, and is structurally unstable in an oxidized state. The structure of soya cerebroside I is stabilized by the presence of polyphenols having a strong antioxidant effect. Since it has a structure in which the 6-position of glucose of soya cerebroside I and the 3-position of cyanidin are combined, a feature that soya cerebroside I is stably maintained arises.

On the other hand, polyphenols are poorly absorbed from the intestinal tract because the intestinal epithelial cells that exhibit absorption have the property of absorbing highly lipophilic substances. Although cyanidin also has low absorbability from the intestinal tract, the soya cerebroside I derivative referred to here increases cyanidin absorption because of its increased fat solubility.

In addition, the tyrosinase inhibitory action has a mechanism in which the soy cerebroside I site of the soya cerebroside I derivative binds to the hydrophobic region of the active center of tyrosinase, and the cyanidin site protects the oxidation of tyrosinase and suppresses melanin production. The soya cerebroside I site and the cyanidin site of the soya cerebroside I derivative are necessary. In addition, the SH group of L-type cysteine exhibits a reducing action, thereby eliminating various oxidative stresses.

Furthermore, the three-dimensional positional relationship between the soya cerebroside I site and the cyanidin site is such that the soya cerebroside I site binds to the hydroxyl group at position 1 of glucose and the cyanidin site binds to the hydroxyl group at position 6 of glucose. It is necessary for suppression of tyrosinase activity.

In addition, by binding three cysteines, various oxidants and adverse reactions can be prevented, the structure is kept stable, and the skin cells, adipose tissue, stromal tissue, fibroblasts and in the cell membrane It is preferable because it easily infiltrates and penetrates to the basal layer of the skin and the tyrosinase inhibitory action is sustained.

When the soya cerebroside I derivative is ingested and absorbed in excess, the excess is degraded by esterase in the blood and decomposed into soya cerebroside I, cyanidin, and cysteine, and each substance has already been confirmed to be safe. Therefore, the soya cerebroside I derivative is also highly safe.

The target soya cerebroside I derivative is obtained by subjecting a fermented product obtained by adding natto to fermented pulverized longan seeds and pulverized soybeans to an alkaline reduction treatment.

Longan has the scientific name Euphonia longana and is a fruit also called longan, longan or longan. This is a tree belonging to the genus Longangan, which is native to India, and currently lives in Southeast Asia such as Thailand and Malaysia, South America such as tropical America and Brazil, Japan, Taiwan and China. The pulp is colorless and translucent jelly. It is sweet and used as a fruit.

Dried longan is used as a medicinal dish and herbal medicine mainly in Southeast Asia. Longan fruit contains black to dark purple seeds. Longan seeds are also rich in dietary experience in Southeast Asia as a folk medicine.

However, not all longan seeds are used as folk medicine, and most of them are discarded, which is a burden on farmers as agricultural waste. Extracting or refining this seed as a raw material is preferable from the viewpoint of effectively utilizing waste and reducing the amount of waste.

First, Longan seeds are crushed. In the pulverization process, free mill, super free mill, sample mill, goblin, super clean mill, micros manufactured by Nara Machinery Co., Ltd. as a pulverizer, a small vacuum dryer manufactured by Toyo Riko as a vacuum dryer, Matsui Co., Ltd. A small vacuum heat transfer dryer DPTH-40 manufactured by AKM, Kyushu Technos Co., Ltd., Clean Dry VD-7, VD-20, and the like are used.

The soybean used here may be any of Japan, China, the United States, or Brazil, and a genetically modified product can also be used. It is preferable to pulverize this soybean with the above-mentioned pulverizer from the viewpoint of efficient fermentation.

The natto bacteria here are a kind of Bacillus subtilis used for the processing of natto and food. Natto bacteria manufactured by Natto Motopo are preferred because they are suitable for fermentation.

The amount of each additive relating to the fermentation is preferably 1 to 4 weights for soybean ground and 1 to 0.001 to 0.05 weight for Bacillus natto per 1 weight of ground longan seeds. The fermentation is preferably carried out in a clean culture tank, and the materials are preferably mixed in advance with tap water or purified water.

This fermentation is heated to 34 to 56 ° C., and the fermentation is performed for 28 to 72 hours. After fermentation, in order to efficiently carry out the following alkali reduction, it is diluted with tap water.

Through this fermentation process, soya cerebroside I and cyanidin in longan seeds are ester-linked by transesterase of Bacillus natto.

The obtained fermentation broth is alkali reduced. In the alkali reduction step, an alkali reduction device or an alkali reduction water conditioner is used. For example, a continuous generator of alkaline reduced water / strongly oxidized water “Protech ATX-501” manufactured by Zemaitis, an alkali reduced water manufacturing apparatus “Techno Super 502” manufactured by NCI, “Mineria CE-212” manufactured by Marthaka, manufactured by Crescent Devices such as “Acura Blue” and “Mineral Reduction Water Conditioner“ Genki no Water ”” manufactured by Nippon Kosen Research Co., Ltd. are preferable. The fermented product is alkali-reduced by these devices.

By this alkali reduction step, the bond between soya cerebroside I and cyanidin is reduced, and at the same time, the hydroxyl group is reduced and stabilized. Further, the fatty acid side chain of soya cerebroside I that has been oxidized by fermentation is reduced to maintain a stable structure.

The desired soya cerebroside I derivative is obtained as a liquid. The soya cerebroside I derivative can be extracted with a vegetable oil or a highly fat-soluble solvent such as ethanol, hexane, chloroform, or butylene glycol described below.

Vegetable oils include palm oil, palm oil, soybean oil, olive oil, rapeseed oil, rice oil, germ oil, corn oil, safflower oil, linseed oil, almond oil, sesame oil, cacao oil, canola oil, grape seed oil, and sesame oil Wheat germ oil, rice bran oil, safflower oil, perilla oil, tea oil, camellia oil, pumpkin seed oil, peanut oil, grape oil, hazelnut oil, cottonseed oil, peanut oil are used.

In the case of extraction, extraction by stirring is preferable, the stirring temperature is preferably 20 to 50 ° C., and the stirring time is preferably 1 to 6 hours.

After the stirring, it is preferable to collect a solvent layer separated into an upper layer and remove the water. In order to remove moisture, a dryer such as TGD-250LF2 manufactured by Toyo Giken is used.

The soya cerebroside I derivative thus isolated is absorbed into the body, and then the excess amount is decomposed by digestive enzymes such as esterase and further metabolized in the liver. Therefore, safety is high and there are few side effects.

It is preferable to separate and purify the desired soya cerebroside I derivative from the alkali reduced product from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin.

As the separation carrier or resin, porous polysaccharide, silicon oxide compound, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymer, etc., whose surface is coated, are used. It is preferred to separate and purify with a suitable separation solvent to remove the organic solvent to obtain the desired soya cerebroside I derivative.

As the solvent for separation, methanol, ethanol, chloroform, hexane, ethyl acetate, benzene, ether, and the like are used. Of these, ethanol for food processing or ethanol containing water is preferable because of its high use range.

The soya cerebroside I derivative thus obtained is obtained as a liquid or powder.

The obtained soya cerebroside I derivative is used for pharmaceuticals, food preparations, cosmetics, hygiene tools, clothing, fibers and the like.

As a pharmaceutical, it is used as a whitening cosmetic agent for suppressing tyrosinase, a skin cancer inhibitor, a local stain-improving pharmaceutical, and the like. In addition, it is also used as a medicine intended for whitening as a veterinary medicine used for animals and a medicine used for pets.

When used as a food preparation, it is used for stains and whitening foods, supplements and drinks.

The cosmetics are used as whitening cosmetics, quasi-drugs and massage oils for the purpose of improving and preventing spots and freckles resulting from tyrosinase inhibitory action.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. In addition, the following description is an example and it can implement by changing a form.

First, the soya cerebroside I derivative obtained by fermentation will be described.

Longan seeds grown in Chiang Mai Province, Thailand were used as raw materials. First, seeds were collected from Longan, washed with water and dried. This was pulverized with a pulverizer (DM-6 manufactured by Nakayama Technical Research Institute).

1 kg of this pulverized Longan seed was put in a clean fermentation tank, and 3 L of tap water was added. On the other hand, Chinese soybeans were washed with water and then heated to 39 ° C. for 90 minutes and pulverized to obtain 2 kg of pulverized soybeans. 1.8 kg of this pulverized soybean was put into the fermentation tank.

To this, 16 g of natto bacteria manufactured by Natto Motopo was added. Fermentation was carried out at a temperature of 40 ° C. with stirring for 40 hours.

3 L of tap water was added to the tank after the fermentation. This was made into a fermented product.

This fermentation broth was subjected to a filter with diatomaceous earth and filtered. The obtained filtrate was subjected to Cellular Kiss (manufactured by Zenon Co., Ltd.), and subjected to alkali reduction by being subjected to an alkali reduction device (alkaline reduced water / strongly oxidized water continuous generator “Protech ATX-501” manufactured by Zemitis).

The alkali reduced product thus obtained was subjected to a lyophilizer manufactured by FP Japan, and 603 g of the desired soya cerebroside I derivative was obtained as a powder. This was designated as Sample 1.

Hereinafter, a purified product of the soya cerebroside I derivative will be described.

100 g of the soya cerebroside I derivative obtained in Example 1 was suspended in 500 mL of ethanol and applied to a column packed with 1.2 kg of Mitsubishi Chemical Diaion. This was washed with 2000 mL of water containing 5% ethanol. Further, after washing with 2000 mL of 10% ethanol-containing water, elution was performed with 800 mL of 50% ethanol-containing water, and then 3 L of 80% ethanol-containing water was flowed, and this fraction was collected.

After subjecting this fraction to a vacuum dryer to distill off ethanol and water, 11 g of purified product of soya cerebroside I derivative was obtained with a freeze dryer manufactured by Nippon FD. This was used as the sample of Example 2.

Below, the identification test of a soya cerebroside I derivative is demonstrated.
(Test Example 1)

The soya cerebroside I derivative obtained in Example 2 obtained as described above was dissolved in purified ethanol, analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation), and further a nuclear magnetic resonance apparatus (NMR And Bruker, AC-250).

As a result, from the sample of Example 2, soya cerebroside I and cyanidin were detected as soya cerebroside I derivatives, and the binding position thereof was such that the 3-position of cyanidin was bound to the 6-position of glucose of soya cerebroside I, Cysteine was bound to the 3rd and 4th positions. All cysteines were in L form.

Further, from the identification test using HPLC, the same soya cerebroside I derivative as described above was also detected in the sample 1 of Example 1.

Below, the test of the tyrosinase inhibitory effect of a soya cerebroside I derivative is demonstrated.

(Test Example 2)
0.5 ml of a sample solution obtained by dissolving the above-described specimen in a tyrosine solution (0.05% concentration), 0.7 ml of a 60 microg / ml solution of tyrosinase solution (manufactured by Sigma), and phosphate buffer (pH 6. 8) 1.8 ml was mixed.

This was reacted at 37 ° C. for 1 hour, and the absorbance A1 at 475 nm was measured. The absorbance A1 was proportional to the concentration of melanin produced from tyrosine.

The same operation was performed for the case where no sample was added, and the absorbance A0 at 475 nm was measured.

The tyrosinase inhibitory action was calculated by the following formula: [tyrosinase inhibitory action (%) = (1−A1 / A0) × 100].

The inhibitory effect was measured by changing the concentration of the sample solution stepwise, and the sample concentration (IC50 value) at which the inhibition rate was 50% was determined by the least square method.

As control substances, arbutin, bakuchiol, vitamin C, hydrogen peroxide, hydroquinone, catechol and heather extract, which are existing tyrosinase inhibitors, were used.

As a result, the IC50 value of the soya cerebroside I derivative obtained in Example 1 was 0.2 microg / ml. The IC50 value of the soya cerebroside I derivative obtained in Example 2 was 0.03 microg / ml.

On the other hand, the IC50 value of arbutin is 6.7 microg / ml, the IC50 value of bakuchiol is 13.6 microg / ml, the IC50 value of vitamin C is 25.6 microg / ml, and the IC50 value of hydrogen peroxide is 11.2 microg / ml, IC50 value of hydroquinone was 6.7 microg / ml, IC50 value of catechol was 5.8 microg / ml, IC50 value of heath extract was 35.5 microg / ml .

From these results, the soya cerebroside I derivative obtained in Example 1 was about 100 times as much as vitamin C, and the soya cerebroside I derivative obtained in Example 2 was a significant tyrosinase inhibitory action over 1000 times that of vitamin C. Was presented.

Examples of cosmetics comprising soya cerebroside I derivatives will be described below.

In a clean cosmetic stainless steel mixer, 10 g of polyethylene glycol monostearate, 20 g of oleophilic glyceryl monostearate, 40 g of horse oil ester and 60 g of oleic acid were heated and dissolved.

To the obtained solution, 100 g of the sample 1 obtained in Example 1 and 900 g of purified water were added. These were dispersed and then cooled to obtain a milky lotion as a cosmetic product. This was designated as Sample 3 of Example 3. As a control cosmetic, an emulsion excluding only the powder of Sample 1 obtained in Example 1 was prepared.

Also, 100 g of vitamin C and 900 g of purified water were added to obtain a control cosmetic containing vitamin C. This was used as a comparative example.

Below, the test example evaluated about the effect and side effect of cosmetics is shown.
(Test Example 4)

For each half of 20 healthy women aged 33 to 69, 7 g of the emulsion of Example 3 or a comparative example containing vitamin C was applied to the right half of the face for 14 days. An emulsion excluding the sample 1 of Example 1 was applied to the left half of the face.

Before application and on the 14th day of application, the amount of spots on the left and right sides of the face was counted using a cosmetic full face photographing apparatus (facial stage) DM-3.

As a result, the amount of the stain on the right half of the face to which the cosmetic containing the soya cerebroside I derivative of Example 3 was applied was 24% before the application, and a decrease in the stain was observed. On the other hand, the left half of the control was 122% compared to before application.

Further, in the comparative example of the vitamin C-containing cosmetic, the stain was reduced to 78% as compared with before application. On the other hand, the left half of the control was 119% compared to before application.

As a result, the cosmetic containing the soya cerebroside I derivative exhibited a stain-preventing action about four times as strong as that of vitamin C. Since vitamin C has a whitening effect that is also used as a pharmaceutical product, it was concluded that the soya cerebroside I derivative exhibits a remarkable stain-preventing action.

Moreover, the feeling of use of the cosmetic containing soya cerebroside I derivative was good.

Furthermore, no side effects of the cosmetic containing soya cerebroside I derivative were observed, and the safety of this cosmetic was confirmed.

The soya cerebroside I derivative having a tyrosinase inhibitory action according to the present invention is suitable for the prevention and improvement of spots and freckles and has few side effects, and thus contributes to the development of the beauty industry.

Moreover, the soya cerebroside I derivative which has a tyrosinase inhibitory action which is the present invention improves the quality of life of people who seek beautiful skin.

Furthermore, since longan seeds are discarded, it promotes the effective use of agricultural resources, reduces the amount of waste, and contributes to the development of agriculture through longan cultivation.

Claims (1)

A soya cerebroside I derivative represented by the following formula (1) having a tyrosinase inhibitory action obtained by subjecting a fermented product obtained by adding natto to fermented pulverized longan seeds and fermented soybeans to an alkali.